I propose to develop the potential of the SV40 genetic system so that it may become a useful probe into eucaryotic recombination mechanisms. As a basis for those studies, I will carry out fine-structure genetic analysis of SV40, using a novel two-step approach that relies on in vitro construction of a presumptive intermediate in SV40 recombination in order to bypass the apparent rate-limiting step in vivo. This combined in vitro-in vivo approach to recombination should permit fine-structure genetic analysis of SV40 that rivals any procaryotic system; the approach lends itself to 3-factor as well as 2-factor crosses, has a resolving power of 0.01% recombination per nucleotide, should not require a mapping function to relate percent recombination to genetic distance, and is expected to yield intermutation distances directly in terms of nucleotides. Within this proposal I outline a simple in vitro method for constructing the multiple mutants of SV40 that are necessary for three-factor mapping. I also indicate how three-factor crosses can be used to evaluate the importance of branch migration and heteroduplex formation in recombination. In addition I propose to use in vitro-constructed SV40 heteroduplexes to assess the efficiency, symmetry, and independence of cellular repair of mismatched bases. (Mismatch repair is an integral concept in current models of recombination, specifically with regard to gene conversion.) Finally, I propose to screen cells that are defective in repair of UV damage for any that are mismatch-repair and/or recombination deficient. Taken together, these studies constitute the beginning of a molecular characterization of recombination in cultured mammalian cells.